RNAi INHIBITION OF CTGF FOR TREATMENT OF OCULAR DISORDERS

ABSTRACT

RNA interference is provided for inhibition of connective tissue growth factor mRNA expression in ocular disorders involving CTGF expression. Ocular disorders involving aberrant CTGF expression include glaucoma, macular degeneration, diabetic retinopathy, choroidal neovascularization, proliferative vitreoretinopathy and wound healing. Such disorders are treated by administering interfering RNAs of the present invention.

The present application is a divisional of U.S. patent application Ser.No. 11/313,200 filed on Dec. 19, 2005 (now allowed), which claimsbenefit to U.S. Provisional Patent Application Ser. No. 60/638,705 filedDec. 23, 2004.

FIELD OF THE INVENTION

The present invention relates to the field of interfering RNAcompositions for inhibition of expression of connective tissue growthfactor (CTGF) in ocular disorders.

BACKGROUND OF THE INVENTION

Most ocular disorders are associated with cellular processes includingcell proliferation, survival, migration, differentiation, andangiogenesis. CTGF is a secreted cytokine and a central mediator inthese cellular processes. In particular, CTGF is known to increaseextracellular matrix production primarily via increased deposition ofcollagen I and fibronectin. Overexpression of CTGF has been implicatedas a major causative factor in conditions such as scleroderma,fibroproliferative diseases, and scarring in which there is anoveraccumulation of extracellular matrix components.

An overaccumulation of extracellular matrix materials in the region ofthe trabecular meshwork (TM) is a hallmark of many forms of glaucoma;such increases are believed to lead to increased resistance to aqueousoutflow and, therefore, elevated intraocular pressures. InternationalPatent Application No. PCT/US2003/012521 to Fleenor et al. publishedNov. 13, 2003 as WO 03/092584 and assigned to Alcon, Inc. describes theelevated presence of CTGF mRNA in glaucomatous TM cells vs. normal TMcells. Thus, it is believed that CTGF plays a role in extracellularmatrix production by the trabecular meshwork cells.

Macular degeneration is the loss of photoreceptors in the portion of thecentral retina, termed the macula, responsible for high-acuity vision.Degeneration of the macula is associated with abnormal deposition ofextracellular matrix components in the membrane between the retinalpigment epithelium and the vascular choroid. This debris-like materialis termed drusen. Drusen is observed using a funduscopic eyeexamination. Normal eyes may have maculas free of drusen, yet drusen maybe abundant in the retinal periphery. The presence of soft drusen in themacula, in the absence of any loss of macular vision, is considered anearly stage of AMD.

Choroidal neovascularization commonly occurs in macular degeneration inaddition to other ocular disorders and is associated with proliferationof choroidal endothelial cells, overproduction of extracellular matrix,and formation of a fibrovascular subretinal membrane. Retinal pigmentepithelium cell proliferation and production of angiogenic factorsappears to effect choroidal neovascularization.

Diabetic retinopathy is an ocular disorder that develops in diabetes dueto thickening of capillary basement membranes and lack of contactbetween pericytes and endothelial cells of the capillaries. Loss ofpericytes increases leakage of the capillaries and leads to breakdown ofthe blood-retina barrier.

Proliferative vitreoretinopathy is associated with cellularproliferation of cellular and fibrotic membranes within the vitreousmembranes and on the surfaces of the retina. Retinal pigment epitheliumcell proliferation and migration is common with this ocular disorder.The membranes associated with proliferative vitreoretinopathy containextracellular matrix components such as collagen types I, II, and IV andfibronectin, and become progressively fibrotic.

Wound healing disorders may lead to severe ocular tissue damage viaactivation of inflammatory cells, release of growth factors andcytokines, proliferation and differentiation of ocular cells, increasedcapillary permeability, alterations in basement membrane matrixcomposition, increased deposition of extracellular matrix, fibrosis,neovascularization, and tissue remodeling.

Overexpression of CTGF therefore has been implicated as a majorcausative factor in these ocular disorders. Current therapies do notdirectly address the pathogenic mechanism of these disorders.

SUMMARY OF THE INVENTION

The present invention is directed to interfering RNAs that target CTGFmRNA and thereby interfere with CTGF mRNA expression. The interferingRNAs of the invention are useful for treating CTGF-related oculardisorders such as glaucoma, macular degeneration, diabetic retinopathy,choroidal neovascularization, proliferative vitreoretinopathy andaberrant wound healing.

An embodiment of the present invention provides a method of attenuatingexpression of connective tissue growth factor mRNA in an eye of asubject. The method comprises administering to the eye of the subject acomposition comprising an effective amount of interfering RNA such asdouble-stranded (ds) siRNA or single-stranded (ss) siRNA having a lengthof 19 to 49 nucleotides and a pharmaceutically acceptable carrier.

The double stranded siRNA comprises a sense nucleotide sequence, anantisense nucleotide sequence and a region of at least near-perfectcontiguous complementarity of at least 19 nucleotides. Further, theantisense sequence hybridizes under physiological conditions to aportion of mRNA corresponding to SEQ ID NO:1 (the sense strand sequenceof DNA for connective tissue growth factor for humans, GenBank referenceno. NM_(—)001901), and has a region of at least near-perfect contiguouscomplementarity of at least 19 nucleotides with the hybridizing portionof mRNA corresponding to SEQ ID NO:1. The administration of such acomposition attenuates the expression of connective tissue growth factormRNA of the eye of the subject.

The single-stranded siRNA has a length of 19 to 49 nucleotides,hybridizes under physiological conditions to a portion of mRNAcorresponding to SEQ ID NO:1 beginning at nucleotide 379, 691, 801, 901,932, 937, 969, 986, 1119, 1170, 1201, 1346, 1473, 1478, 1481, 1488,1626, 1660, or 1666, and has a region of at least near-perfectcomplementarity with the hybridizing portion of mRNA corresponding toSEQ ID NO:1.

In an embodiment of the invention, the antisense sequence of adouble-stranded interfering RNA is designed to target a nucleotidesequence of mRNA corresponding to SEQ ID NO:1 beginning at or comprisingnucleotide 379, 691, 801, 901, 932, 937, 969, 986, 1119, 1170, 1201,1346, 1473, 1478, 1481, 1488, 1626, 1660, or 1666.

A further embodiment of the invention is a method of treating aconnective tissue growth factor-associated ocular disorder in a subjectin need thereof. The method comprises administering to the eye of thesubject a composition comprising an effective amount of interfering RNAhaving a length of 19 to 49 nucleotides and a pharmaceuticallyacceptable carrier, the interfering RNA comprising a sense nucleotidesequence, an antisense nucleotide sequence, and a region of at leastnear-perfect contiguous complementarity of at least 19 nucleotides. Theantisense sequence hybridizes under physiological conditions to aportion of mRNA corresponding to SEQ ID NO:1, and has a region of atleast near-perfect contiguous complementarity of at least 19 nucleotideswith the hybridizing portion of mRNA corresponding to SEQ ID NO:1. Theconnective tissue growth factor-associated ocular disorder is treatedthereby.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows SITOX™ data demonstrating that transfection efficiency oftrabecular meshwork cells was not rate-limiting when taken together withdata of FIG. 1B. GTM3 cells were transfected with SITOX™ (Dharmacon)transfection control. After 24 hr, trypan blue exclusion was used todetermine the number of viable cells remaining in the SITOX™ culture,which reflects the relative transfection efficiency. Open bars: notransfection; Solid bars: with SITOX™.

FIG. 1B shows a SIGLO™ image of siRNA uptake in GTM3 cells demonstratingthat transfection efficiency was not rate-limiting when taken togetherwith data of FIG. 1A. GTM3 cells were transfected with SIGLO™ siRNA(Dharmacon) using LIPOFECTAMINE 2000™. SIGLO™ siRNA uptake wasdetermined after 24 hr using fluorescence microscopy (red irregularshapes). Individual cell nuclei were identified by DAPI(4′,6-diamidino-2-phenylindole), a stain for double stranded DNA (blueround areas). As the data of FIG. 1A and the image of FIG. 1B show,nearly all cells were either dead (SITOX™) or fluorescent (SIGLO™).

FIG. 2A is a schematic showing the CTGF gene exon (boxes) and intron(lines) structure and location of siRNAs S1, S2, and S3 and QPCRprimer/probe sets Q1 and Q2 in relation to the GenBank CTGF sequenceNM_(—)001901, the sequence of which is provided as SEQ ID NO:1. Thesequences of the siRNAs and primer/probe sets are provided in Example 1.

FIG. 2B shows QPCR amplification of CTGF mRNA using the exon 5primer/probe set Q2. Using the S1 and S4 siRNAs, no significantknock-down of the CTGF mRNA levels was detected.

FIG. 2C shows QPCR amplification of CTGF mRNA using the exon 4/5spanning primer/probe set Q1. Knock-down of CTGF mRNA was observed byeach of the siRNAs and an ˜90% knock-down was observed with the S2siRNA.

FIG. 3 shows a titration study in which various concentrations of the S2siRNA were tested for efficacy of knock-down of CTGF mRNA levels. CTGFmRNA knock-down was assessed by QPCR amplification using primer/probeset Q1. An IC₅₀ of ˜2.5 nM was observed in GTM3 cells after 24 hourtreatment with 0, 1, 3, 10, 30, and 100 nM S2 siRNA as described inExample 1.

DETAILED DESCRIPTION OF THE INVENTION

RNA interference, termed “RNAi,” is a method for reducing the expressionof a target gene that is effected by small single- or double-strandedRNA molecules. Interfering RNAs include small interfering RNAs, eitherdouble-stranded or single-stranded (ds siRNAs or ss siRNAs), microRNAs(miRNAs), small hairpin RNAs (shRNAs), and others. While not wanting tobe bound by theory, RNA interference appears to occur in vivo with thecleavage of dsRNA precursors into small RNAs of about 20 to 25nucleotides in length. Cleavage is accomplished by RNaseIII-RNA helicaseDicer.

The “sense” strand of an siRNA, i.e., the strand that has exactly thesame sequence as a target mRNA sequence, is removed, leaving the“antisense” strand which is complementary to the target mRNA to functionin reducing expression of the mRNA. The antisense strand of the siRNAappears to guide a protein complex known as RISC(RNA-induced silencingcomplex) to the mRNA, which complex then cleaves the mRNA by theArgonaute protein of the RISC, thereby reducing protein production bythat mRNA. Interfering RNAs are catalytic and reduction in expression ofmRNA can be achieved with substoichiometric amounts of interfering RNAsin relation to mRNA. Reduction in mRNA expression may also occur viatranscriptional and translational mechanisms.

The present invention relates to the use of interfering RNA forinhibition of expression of connective tissue growth factor (CTGF) inocular disorders. According to the present invention, tissues of theeye, in particular, trabecular meshwork cells of the eye, carry outsiRNA silencing, and exogenously provided siRNAs effect silencing.Further, aspects of the present invention have determined that, whenusing a PCR-based approach to determine the efficacy of siRNAknock-down, the PCR amplification primers should be designed toencompass the siRNA targeting sequence to accurately measure silencing.

Nucleic acid sequences cited herein are written in a 5′ to 3′ directionunless indicated otherwise. The term “nucleic acid,” as used herein,refers to either DNA or RNA or a modified form thereof comprising thepurine or pyrimidine bases present in DNA (adenine “A,” cytosine “C,”guanine “G,” thymine “T”) or in RNA (adenine “A,” cytosine “C,” guanine“G,” uracil “U”). Interfering RNAs provided herein may comprise “T”bases, particularly at 3′ ends, even though “T” bases do not naturallyoccur in RNA. “Nucleic acid” includes the terms “oligonucleotide” and“polynucleotide” and can refer to a single stranded molecule or a doublestranded molecule. A double stranded molecule is formed by Watson-Crickbase pairing between A and T bases, C and G bases, and A and U bases.The strands of a double stranded molecule may have partial, substantialor full complementarity to each other and will form a duplex hybrid, thestrength of bonding of which is dependent upon the nature and degree ofcomplementarity of the sequence of bases. A mRNA sequence is readilydetermined by knowing the sense or antisense strand sequence of DNAencoding therefor. For example, SEQ ID NO:1 provides the sense strandsequence of DNA corresponding to the mRNA for connective tissue growthfactor. The sequence of mRNA is identical to the sequence of the sensestrand of DNA with the “T” bases replaced with “U” residues. Therefore,the mRNA sequence of connective tissue growth factor is known from SEQID NO:1.

Connective tissue growth factor mRNA: The GenBank database of theNational Center for Biotechnology Information at ncbi.nlm.nih.govprovides the corresponding DNA sequence for the messenger RNA of humanconnective tissue growth factor as reference no. NM_(—)001901, providedbelow as SEQ ID NO:1. The coding sequence for connective tissue growthfactor is from nucleotides 146-1195.

SEQ ID NO: 1: 1 tccagtgacg gagccgcccg gccgacagcc ccgagacgac agcccggcgcgtcccggtcc 61 ccacctccga ccaccgccag cgctccaggc cccgcgctcc ccgctcgccgccaccgcgcc 121 ctccgctccg cccgcagtgc caaccatgac cgccgccagt atgggccccgtccgcgtcgc 181 cttcgtggtc ctcctcgccc tctgcagccg gccggccgtc ggccagaactgcagcgggcc 241 gtgccggtgc ccggacgagc cggcgccgcg ctgcccggcg ggcgtgagcctcgtgctgga 301 cggctgcggc tgctgccgcg tctgcgccaa gcagctgggc gagctgtgcaccgagcgcga 361 cccctgcgac ccgcacaagg gcctcttctg tgacttcggc tccccggccaaccgcaagat 421 cggcgtgtgc accgccaaag atggtgctcc ctgcatcttc ggtggtacggtgtaccgcag 481 cggagagtcc ttccagagca gctgcaagta ccagtgcacg tgcctggacggggcggtggg 541 ctgcatgccc ctgtgcagca tggacgttcg tctgcccagc cctgactgccccttcccgag 601 gagggtcaag ctgcccggga aatgctgcga ggagtgggtg tgtgacgagcccaaggacca 661 aaccgtggtt gggcctgccc tcgcggctta ccgactggaa gacacgtttggcccagaccc 721 aactatgatt agagccaact gcctggtcca gaccacagag tggagcgcctgttccaagac 781 ctgtgggatg ggcatctcca cccgggttac caatgacaac gcctcctgcaggctagagaa 841 gcagagccgc ctgtgcatgg tcaggccttg cgaagctgac ctggaagagaacattaagaa 901 gggcaaaaag tgcatccgta ctcccaaaat ctccaagcct atcaagtttgagctttctgg 961 ctgcaccagc atgaagacat accgagctaa attctgtgga gtatgtaccgacggccgatg 1021 ctgcaccccc cacagaacca ccaccctgcc ggtggagttc aagtgccctgacggcgaggt 1081 catgaagaag aacatgatgt tcatcaagac ctgtgcctgc cattacaactgtcccggaga 1141 caatgacatc tttgaatcgc tgtactacag gaagatgtac ggagacatggcatgaagcca 1201 gagagtgaga gacattaact cattagactg gaacttgaac tgattcacatctcatttttc 1261 cgtaaaaatg atttcagtag cacaagttat ttaaatctgt ttttctaactgggggaaaag 1321 attcccaccc aattcaaaac attgtgccat gtcaaacaaa tagtctatcttccccagaca 1381 ctggtttgaa gaatgttaag acttgacagt ggaactacat tagtacacagcaccagaatg 1441 tatattaagg tgtggcttta ggagcagtgg gagggtacca gcagaaaggttagtatcatc 1501 agatagctct tatacgagta atatgcctgc tatttgaagt gtaattgagaaggaaaattt 1561 tagcgtgctc actgacctgc ctgtagcccc agtgacagct aggatgtgcattctccagcc 1621 atcaagagac tgagtcaagt tgttccttaa gtcagaacag cagactcagctctgacattc 1681 tgattcgaat gacactgttc aggaatcgga atcctgtcga ttagactggacagcttgtgg 1741 caagtgaatt tcctgtaaca agccagattt tttaaaattt atattgtaaatattgtgtgt 1801 gtgtgtgtgt gtgtatatat atatatatat gtacagttat ctaagttaatttaaagttgt 1861 ttgtgccttt ttatttttgt ttttaatgct ttgatatttc aatgttagcctcaatttctg 1921 aacaccatag gtagaatgta aagcttgtct gatcgttcaa agcatgaaatggatacttat 1981 atggaaattc tctcagatag aatgacagtc cgtcaaaaca gattgtttgcaaaggggagg 2041 catcagtgtc cttggcaggc tgatttctag gtaggaaatg tggtagctcacgctcacttt 2101 taatgaacaa atggccttta ttaaaaactg agtgactcta tatagctgatcagttttttc 2161 acctggaagc atttgtttct actttgatat gactgttttt cggacagtttatttgttgag 2221 agtgtgacca aaagttacat gtttgcacct ttctagttga aaataaagtatattttttct 2281 aaaaaaaaaa aaaaacgaca gcaacggaat tc.Equivalents of the above cited CTGF mRNA sequence are alternative spliceforms, allelic forms, or a cognate thereof. A cognate is a connectivetissue growth factor mRNA from another mammalian species that ishomologous to SEQ ID NO:1. CTGF nucleic acid sequences related to SEQ IDNO:1 are those having GenBank accession numbers AK092280, AK125220,AY395801, AY550024, BT019794, BT019795, CR541759, M92934, U14750, andX78947, and the sequence of SEQ ID NO:1 of U.S. Pat. No. 5,585,270,incorporated by reference herein.

Attenuating expression of an mRNA: The phrase, “attenuating expressionof an mRNA,” as used herein, means administering an amount ofinterfering RNA to effect a reduction of the full mRNA transcript levelsof a target gene in a cell, thereby decreasing translation of the mRNAinto protein as compared to a control RNA having a scrambled sequence.The reduction in expression of the mRNA is commonly referred to as“knock-down” of mRNA. Knock-down of expression of an amount of betweenand including an amount of 50% and 100% is contemplated by embodimentsherein. However, it is not necessary that such knock-down levels beachieved for purposes of the present invention. Further, two sets ofinterfering RNAs may be mildly effective at knock-down individually,however, when administered together may be significantly more effective.In one embodiment, an individual ds siRNA is effective at knock-down atan amount of at least up to 70%. In another embodiment, two or more dssi RNAs are together effective at knock-down at an amount of at least upto 70%.

Knock-down is commonly measured by determining the mRNA levels byQuantitative Polymerase Chain Reaction (QPCR) amplification or bydetermining protein levels by Western Blot or enzyme linkedimmunosorbent assay (ELISA). Analyzing the protein level provides anassessment of both mRNA degradation by the RNA Induced Silencing Complex(RISC) as well as translation inhibition. Further techniques formeasuring knock-down include RNA solution hybridization, nucleaseprotection, Northern hybridization, reverse transcription, geneexpression monitoring with a microarray, antibody binding,radioimmunoassay, and fluorescence activated cell analysis. A furthermethod of measurement includes overexpressing TGFβ2 which induces CTGF,adding back CTGF siRNA, and then measuring CTGF mRNA/protein knockdownby any of the above-cited methods.

Inhibition of CTGF is also inferred in a human or mammal by observing animprovement in an ocular disorder. For example, in age related maculardegeneration a slowing or reversal of vision loss indicates aninhibition of CTGF and silencing of CTGF mRNA in glaucoma patients leadsto lowered intraocular pressure and a delay or prevention of the onsetof symptoms in a subject at risk for developing glaucoma.

Interfering RNA of embodiments of the invention act in a catalyticmanner, i.e., interfering RNA is able to effect inhibition of targetmRNA in substoichiometric amounts. As compared to antisense therapies,significantly less interfering RNA is required to provide a therapeuticeffect.

Double-stranded interfering RNA: Double stranded interfering RNA (alsoreferred to as ds siRNA), as used herein, has a sense nucleotidesequence and an antisense nucleotide sequence, the sense and antisensesequence comprising a region of at least near-perfect contiguouscomplementarity of at least 19 nucleotides. The length of theinterfering RNA comprises 19 to 49 nucleotides, and may comprise alength of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49nucleotides. The antisense sequence of the ds siRNA hybridizes underphysiological conditions to a portion of mRNA corresponding to SEQ IDNO:1, and has a region of at least near-perfect contiguouscomplementarity of at least 19 nucleotides with the hybridizing portionof mRNA corresponding to SEQ ID NO:1.

The antisense strand of the siRNA is the active guiding agent of thesiRNA in that the antisense strand binds to a RISC complex within acell, and guides the bound complex to bind with specificity to the mRNAat a sequence complementary to the sequence of the antisense RNA,thereby allowing subsequent cleavage of the mRNA by the bound complex.

Techniques for selecting target sequences for siRNAs are provided byTuschl, T. et al, “The siRNA User Guide,” revised May 6, 2004, availableon the Rockefeller University web site, by Technical Bulletin #506,“siRNA Design Guidelines,” Ambion Inc. at Ambion's web site, by theInvitrogen web site using search parameters of min 35%, max 55% G/Ccontent, and by the Dharmacon web site. The target sequence may belocated in the coding region or a 5′ or 3′ untranslated region of themRNA.

An embodiment of a DNA target sequence for CTGF is present atnucleotides 1488 to 1506 of SEQ ID NO:1:

5′- ggttagtatcatcagatag-3′. SEQ ID NO: 18 nt 1488.A double stranded siRNA of the invention for targeting a correspondingmRNA sequence of SEQ ID NO:18 and having a 3′UU overhang on each strandis:

5′- gguuaguaucaucagauagUU-3′ SEQ ID NO: 25 3′- UUccaaucauaguagucuauc-5′.SEQ ID NO: 26The 3′ overhang may have a number of “U” residues, for example, a numberof “U” residues between and including 2, 3, 4, 5, and 6. The 5′ end mayalso have a 5′ overhang of nucleotides. A double stranded siRNA of theinvention for targeting a corresponding mRNA sequence of SEQ ID NO:18and having a 3′TT overhang on each strand is:

5′- gguuaguaucaucagauagTT-3′ SEQ ID NO: 27 3′- TTccaaucauaguagucuauc-5′.SEQ ID NO: 28The strands of a double-stranded siRNA may be connected by a hairpinloop to form a single stranded siRNA as follows:

 5′- gguuaguaucaucagauagUUNNN\ SEQ ID NO: 29                              N 3′- UUccaaucauaguagucuaucNNNNN/.N is a nucleotide A, T, C, G, U, or a modified form known by one ofordinary skill in the art. The number of nucleotides N is a numberbetween and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9to 11, or the number of nucleotides N is 9.

Table 1 lists examples of CTGF DNA target sequences of SEQ ID NO:1 fromwhich siRNAs of the present invention are designed in a manner as setforth above.

TABLE 1 CTGF Target Sequences fcr siRNAs # of Starting Nucleotide withreference to SEQ ID Target Sequence SEQ ID NO: 1 NO: GGGCCTCTTCTGTGACTTC379 2 CCGACTGGAAGACACGTTT 691 3 CCCGGGTTACCAATGACAA 801 4GGGCAAAAAGTGCATCCGT 901 5 TCCAAGCCTATCAAGTTTGAGCTTT 932 6GCCTATCAAGTTTGAGCTT 937 7 GCATGAAGACATACCGAGCTAAATT 969 8GCTAAATTCTGTGGAGTAT 986 9 GCCATTACAACTGTCCCGGAGACAA 1119 10GGAAGATGTACGGAGACAT 1170 11 GAGAGTGAGAGACATTAACTCATTA 1201 12GCCATGTCAAACAAATAGTCTATCT 1346 13 GGGTACCAGCAGAAAGGTT 1473 14CCAGCAGAAAGGTTAGTAT 1478 15 GCAGAAAGGTTAGTATCAT 1481 16GCAGAAAGGTTAGTATCATCAGATA 1481 17 GGTTAGTATCATCAGATAG 1488 18GGTTAGTATCATCAGATAGCTCTTA 1488 19 GAGACTGAGTCAAGTTGTTCCTTAA 1626 20GCAGACTCAGCTCTGACAT 1660 21 TCAGCTCTGACATTCTGATTCGAAT 1666 22TCCTGTCGATTAGACTGGACAGCTT 1712 23 GCTTGTGGCAAGTGAATTT 1733 24As cited in the examples above, one of skill in the art is able to usethe target sequence information provided in Table 1 to designinterfering RNAs having a length shorter or longer than the sequencesprovided in Table 1 by referring to the sequence position in SEQ ID NO:1and adding or deleting nucleotides complementary or near complementaryto SEQ ID NO:1.

The target RNA cleavage reaction guided by ds or ss siRNAs is highlysequence specific. In general, siRNA containing a sense nucleotidesequence identical to a portion of the target mRNA and an antisenseportion exactly complementary to the sense sequence are siRNAembodiments for inhibition of CTGF mRNA. However, 100% sequencecomplementarity between the antisense strand of siRNA and the targetmRNA is not required to practice the present invention. Thus theinvention allows for sequence variations that might be expected due togenetic mutation, strain polymorphism, or evolutionary divergence. Forexample, siRNA sequences with insertions, deletions, or single pointmutations relative to the target sequence are effective for inhibition.

The antisense sequence of the siRNA has at least near-perfect contiguouscomplementarity of at least 19 nucleotides with the target sequence ofthe mRNA. “Near-perfect,” as used herein, means the antisense sequenceof the siRNA is “substantially complementary to,” and the sense sequenceof the siRNA is “substantially identical” to at least a portion of thetarget mRNA. “Identity,” as known by one of ordinary skill in the art,is the degree of sequence relatedness between nucleotide sequences asdetermined by matching the order of nucleotides between the sequences.In one embodiment, antisense RNA having 80% and between 80% up to 100%complementarity to the target mRNA sequence are considered near-perfectcomplementarity and may be used in the present invention. “Perfect”contiguous complementarity is standard Watson-Crick base pairing ofadjacent base pairs. “At least near-perfect” contiguous complementarityincludes “perfect” complementarity as used herein. Computer methods fordetermining identity or complementarity are designed to provide thegreatest degree of matching of nucleotide sequences, for example, BLASTPand BLASTN (Altschul, S. F., et al. (1990) J. Mol. Biol. 215:403-410),and FASTA.

The target sequence of SEQ ID NO:1 may be in the 5′ or 3′ untranslatedregions of the mRNA as well as in the coding region of the mRNA.

One or both of the strands of double-stranded interfering RNA may have a3′ overhang of from 1 to 6 nucleotides which may be ribonucleotides ordeoxyribonucleotides or a mixture thereof.

The nucleotides of the overhang are not base-paired. In one embodimentof the invention, the interfering ds RNA comprises a 3′ overhang of TTor UU.

The sense and antisense strands of the double stranded siRNA may be in aduplex formation of two single strands as described above or may be asingle molecule where the regions of complementarity are base-paired andare covalently linked by a hairpin or loop so as to form a singlestrand. It is believed that the hairpin is cleaved intracellularly by aprotein termed Dicer to form an interfering RNA of two individualbase-paired RNA molecules.

Interfering RNAs may differ from naturally-occurring RNA by theaddition, deletion, substitution or modification of one or morenucleotides. Non-nucleotide material may be bound to the interferingRNA, either at the 5′ end, the 3′ end, or internally. Such modificationsare commonly designed to increase the nuclease resistance of theinterfering RNAs, to improve cellular uptake, to enhance cellulartargeting, to assist in tracing the interfering RNA, or to furtherimprove stability. For example, interfering RNAs may comprise a purinenucleotide at the ends of overhangs. Conjugation of cholesterol to the3′ end of the sense strand of a ds siRNA molecule by means of apyrrolidine linker, for example, also provides stability to an siRNA.Further modifications include a 3′ terminal biotin molecule, a peptideknown to have cell-penetrating properties, a nanoparticle, apeptidomimetic, a fluorescent dye, or a dendrimer, for example.

Nucleotides may be modified on their base portion, on their sugarportion, or on the phosphate portion of the molecule and function inembodiments of the present invention. Modifications includesubstitutions with alkyl, alkoxy, amino, deaza, halo, hydroxyl, thiolgroups, or a combination thereof, for example. Nucleotides may besubstituted with analogs with greater stability such as replacing U with2′deoxy-T, or having a sugar modification such as a 2′OH replaced by a2′ amino or 2′ methyl group, 2′methoxyethyl groups, or a 2′-O, 4′-Cmethylene bridge, for example. Examples of a purine or pyrimidine analogof nucleotides include a xanthine, a hypoxanthine, an azapurine, amethylthioadenine, 7-deaza-adenosine and O- and N-modified nucleotides.The phosphate group of the nucleotide may be modified by substitutingone or more of the oxygens of the phosphate group with nitrogen or withsulfur (phosphorothioates).

There may be a region of the antisense siRNA that is not complementaryto a portion of mRNA corresponding to SEQ ID NO:1. Non-complementaryregions may be at the 3′, 5′ or both ends of a complementary region.

Interfering RNAs may be synthetically generated, generated by in vitrotranscription, siRNA expression vectors, or PCR expression cassettes,for example. Interfering RNAs that function well as transfected siRNAsalso function well as siRNAs expressed in vivo.

Interfering RNAs are chemically synthesized using protectedribonucleoside phosphoramidites and a conventional DNA/RNA synthesizerand may be obtained from commercial suppliers such as Ambion Inc.(Austin, Tex.), Invitrogen (Carlsbad, Calif.), or Dharmacon (Lafayette,Colo., USA), for example. Interfering RNAs are purified by extractionwith a solvent or resin, precipitation, electrophoresis, chromatography,or a combination thereof, for example. Alternatively, interfering RNAmay be used with little if any purification to avoid losses due tosample processing.

Interfering RNA may be provided to a subject by expression from arecombinant plasmid using a constitutive or inducible promoter such asthe U6 or H1 RNA pol III promoter, the cytomegalovirus promoter, SP6,T3, or T7 promoter, known to those of ordinary skill in the art. Forexample, the psiRNA™ from InvivoGen (San Diego, Calif.) allowsproduction of siRNAs within cells from an RNA pol III promoter.Interfering RNA expressed from recombinant plasmids may be isolated bystandard techniques.

A viral vector for expression of interfering RNA may be derived fromadenovirus, adeno-associated virus, vaccinia virus, retroviruses(lentiviruses, Rhabdoviruses, murine leukemia virus, for example),herpes virus, or the like, using promoters as cited above, for example,for plasmids. Selection of viral vectors, methods for expressing theinterfering RNA by the vector and methods of delivering the viral vectorare within the ordinary skill of one in the art.

Expression of interfering RNAs is also provided by use of SILENCEREXPRESS™ (Ambion, Austin, Tex.) via expression cassettes (SECs) with ahuman H1, human U6 or mouse U6 promoter by PCR. Silencer expressioncassettes are PCR products that include promoter and terminatorsequences flanking a hairpin siRNA template. Upon transfection intocells, the hairpin siRNA is expressed from the PCR product and inducesspecific silencing.

Hybridization under Physiological Conditions: “Hybridization” refers toa technique where single-stranded nucleic acids (DNA or RNA) are allowedto interact so that hydrogen-bonded complexes called hybrids are formedby those nucleic acids with complementary or near-complementary basesequences. Hybridization reactions are sensitive and selective so that aparticular sequence of interest is identified in samples in which it ispresent at low concentrations. The specificity of hybridization (i.e.,stringency) is controlled by the concentrations of salt or formamide inthe prehybridization and hybridization solutions in vitro, for example,and by the hybridization temperature, and are well known in the art. Inparticular, stringency is increased by reducing the concentration ofsalt, increasing the concentration of formamide, or raising thehybridization temperature.

For example, high stringency conditions could occur at about 50%formamide at 37° C. to 42° C. Reduced stringency conditions could occurat about 35% to 25% formamide at about 30° C. to 35° C. Examples ofstringency conditions for hybridization are provided in Sambrook, J.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. Further examples of stringenthybridization conditions include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing, orhybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC, or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The temperature for hybridization is about 5-10° C. less than themelting temperature (T_(m)) of the hybrid where T_(m) is determined forhybrids between 19 and 49 base pairs in length using the followingcalculation: T_(m)° C.=81.5+16.6(log₁₀[Na+])+0.41 (% G+C)−(600/N) whereN is the number of bases in the hybrid, and [Na+] is the concentrationof sodium ions in the hybridization buffer.

In embodiments of the present invention, an antisense strand of aninterfering RNA that hybridizes with CTGF mRNA in vitro under highstringency conditions will bind specifically in vivo under physiologicalconditions. Identification or isolation of a related nucleic acid thatdoes not hybridize to a nucleic acid under highly stringent conditionsis carried out under reduced stringency.

Single stranded interfering RNA: As cited above, interfering RNAsultimately function as single strands. SS siRNA has been found to effectmRNA silencing, albeit less efficiently than double-stranded RNA.Therefore, embodiments of the present invention also provide foradministration of ss siRNA where the single stranded siRNA hybridizesunder physiological conditions to a portion of mRNA corresponding to SEQID NO:1, and has a region of at least near-perfect contiguouscomplementarity of at least 19 nucleotides with the hybridizing portionof mRNA corresponding to SEQ ID NO:1. The ss siRNA has a length of 19 to49 nucleotides as for the ds siRNA cited above. The ss siRNA has a 5′phosphate or is phosphorylated in situ or in vivo at the 5′ position.The term “5′ phosphorylated” is used to describe, for example,polynucleotides or oligonucleotides having a phosphate group attachedvia ester linkage to the C5 hydroxyl of the 5′ sugar (e.g., the 5′ribose or deoxyribose, or an analog of same). The ss siRNA may have amono-, di-, or triphosphate group.

SS siRNAs are synthesized chemically or via vectors as for ds siRNAs. 5′Phosphate groups may be added via a kinase, or a 5′ phosphate may be theresult of nuclease cleavage of an RNA. Delivery is as for ds siRNAs. Inone embodiment, ss siRNAs having protected ends and nuclease resistantmodifications are administered for silencing. SS siRNAs may be dried forstorage or dissolved in an aqueous solution. The solution may containbuffers or salts to inhibit annealing or for stabilization.

Hairpin interfering RNA: A hairpin interfering RNA is single-strandedand contains both the sense and antisense sequence within the onestrand. For expression by a DNA vector, the corresponding DNAoligonucleotides of at least 19-nucleotides corresponding to the sensesiRNA sequence are linked to its reverse complementary antisensesequence by a short spacer. If needed for the chosen expression vector,3′ terminal T's and nucleotides forming restriction sites may be added.The resulting RNA transcript folds back onto itself to form a stem-loopstructure.

Mode of administration: Interfering RNA may be delivered directly to theeye by ocular tissue injection such as periocular, conjunctival,sub-Tenons, intracameral, intravitreal, sub-retinal, retrobulbar, orintracanalicular injections; by direct application to the eye using acatheter or other placement device such as a retinal pellet, intraocularinsert, suppository or an implant comprising a porous, non-porous, orgelatinous material; by topical ocular drops or ointments; by a slowrelease device in the cul-de-sac or implanted adjacent to the sclera(transsclerahl) or within the eye. Intracameral injection may be throughthe cornea into the anterior chamber to allow the agent to reach thetrabecular meshwork. Intracanalicular injection may be into the venouscollector channels draining Schlemm's canal or into Schlemm's canal.

Subject: A subject in need of treatment for an ocular disorder or atrisk for developing an ocular disorder is a human or other mammal havinga condition or at risk of having a condition associated with expressionor activity of CTGF, i.e., a CTGF-associated ocular disorder. Such anocular disorder may include, for example, glaucoma, maculardegeneration, diabetic retinopathy, choroidal neovascularization,proliferative vitreoretinopathy, wound healing, and conditions withexcessive scarring, with endothelial cell proliferation, orfibroproliferation. Ocular structures associated with such disorders mayinclude the retina, choroid, lens, cornea, trabecular meshwork, rod,cone, ganglia, macula, iris, sclera, aqueous chamber, vitreous chamber,ciliary body, optic disc, papilla, or fovea, for example.

Formulations and Dosage: Pharmaceutical formulations comprise aninterfering RNA, or salt thereof, of the invention up to 99% by weightmixed with a physiologically acceptable ophthalmic carrier medium suchas water, buffer, saline, glycine, hyaluronic acid, mannitol, and thelike.

Interfering RNAs of the present invention are administered as solutions,suspensions, or emulsions. The following are examples of possibleformulations embodied by this invention.

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Hydroxypropylmethylcellulose 0.5 Sodium chloride  .8 BenzalkoniumChloride 0.01 EDTA 0.01 NaOH/HCl qs pH 7.4 Purified water qs 100 mL

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Phosphate Buffered Saline 1.0 Benzalkonium Chloride 0.01 Polysorbate 800.5 Purified water q.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Monobasic sodium phosphate 0.05 Dibasic sodium phosphate 0.15(anhydrous) Sodium chloride 0.75 Disodium EDTA 0.05 Cremophor EL 0.1Benzalkonium chloride 0.01 HCl and/or NaOH pH 7.3-7.4 Purified waterq.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Phosphate Buffered Saline 1.0 Hydroxypropyl-β-cyclodextrin 4.0 Purifiedwater q.s. to 100%

Generally, an effective amount of the interfering RNA of embodiments ofthe invention comprises an intercellular concentration at or near theocular site of from 200 μM to 100 nM, or from 1 nM to 50 nM, or from 5nM to about 25 nM. Topical compositions are delivered to the surface ofthe eye one to four times per day according to the routine discretion ofa skilled clinician. The pH of the formulation is about pH 4-9, or pH4.5 to pH 7.4.

While the precise regimen is left to the discretion of the clinician,interfering RNA may be administered by placing one drop in each eye oneto four times a day, or as directed by the clinician. An effectiveamount of a formulation may depend on factors such as the age, race, andsex of the subject, or the severity of the ocular disorder, for example.In one embodiment, the interfering RNA is delivered topically to the eyeand reaches the trabecular meshwork, retina or optic nerve head at atherapeutic dose thereby ameliorating a CTGF-associated disease process.

Acceptable carriers: An ophthalmically acceptable carrier refers tothose carriers that cause at most, little to no ocular irritation,provide suitable preservation if needed, and deliver one or moreinterfering RNAs of the present invention in a homogenous dosage. Anacceptable carrier for administration of interfering RNA of embodimentsof the present invention include the Mirus TransIT®-TKO siRNATransfection Reagent (Mirus Corporation, Madison, Wis.), LIPOFECTIN®,lipofectamine, OLIGOFECTAMINE™ (Invitrogen, Carlsbad, Calif.),CELLFECTIN®, DHARMAFECT™ (Dharmacon, Chicago, Ill.) or polycations suchas polylysine, liposomes, or fat-soluble agents such as cholesterol.Liposomes are formed from standard vesicle-forming lipids and a sterol,such as cholesterol, and may include a targeting molecule such as amonoclonal antibody having binding affinity for endothelial cell surfaceantigens, for example. Further, the liposomes may be PEGylatedliposomes.

For ophthalmic delivery, an interfering RNA may be combined withopthalmologically acceptable preservatives, co-solvents, surfactants,viscosity enhancers, penetration enhancers, buffers, sodium chloride, orwater to form an aqueous, sterile ophthalmic suspension or solution.Ophthalmic solution formulations may be prepared by dissolving theinhibitor in a physiologically acceptable isotonic aqueous buffer.Further, the ophthalmic solution may include an opthalmologicallyacceptable surfactant to assist in dissolving the inhibitor. Viscositybuilding agents, such as hydroxymethyl cellulose, hydroxyethylcellulose, methylcellulose, polyvinylpyrrolidone, or the like, may beadded to the compositions of the present invention to improve theretention of the compound.

In order to prepare a sterile ophthalmic ointment formulation, theinterfering RNA is combined with a preservative in an appropriatevehicle, such as mineral oil, liquid lanolin, or white petrolatum.Sterile ophthalmic gel formulations may be prepared by suspending theinterfering RNA in a hydrophilic base prepared from the combination of,for example, CARBOPOL®-940 (BF Goodrich, Charlotte, N.C.), or the like,according to methods known in the art for other ophthalmic formulations.VISCOAT® (Alcon Laboratories, Inc., Fort Worth, Tex.) may be used forintraocular injection, for example. Other compositions of the presentinvention may contain penetration enhancing agents such as cremephor andTWEEN® 80 (polyoxyethylene sorbitan monolaureate, Sigma Aldrich, St.Louis, Mo.), in the event the interfering RNA is less penetrating in theeye.

Kits: Embodiments of the present invention provide a kit that includesreagents for attenuating the expression of a CTGF mRNA in a cell. Thekit contains a DNA template that has two different promoters such as aT7 promoter, a T3 promoter or an SP6 promoter, each operably linked to anucleotide sequence that encodes two complementary single-stranded RNAscorresponding to an interfering RNA. RNA is transcribed from the DNAtemplate and is annealed to form a double-stranded RNA effective toattenuate expression of the target mRNA. The kit optionally containsamplification primers for amplifying the DNA sequence from the DNAtemplate and nucleotide triphosphates (i.e., ATP, GTP, CTP and UTP) forsynthesizing RNA. Optionally, the kit contains two RNA polymerases, eachcapable of binding to a promoter on the DNA template and effectingtranscription of the nucleotide sequence to which the promoter isoperably linked, a purification column for purifying single-strandedRNA, such as a size exclusion column, one or more buffers, for example,a buffer for annealing single-stranded RNAs to yield double strandedRNA, and RNAse A or RNAse T for purifying double stranded RNA.

Example 1 Interfering RNA for Silencing CTGF in Trabecular MeshworkCells and Criteria for Measuring Silencing

The present study examines the ability of CTGF interfering RNA toknock-down the levels of endogenous CTGF expression in human trabecularmeshwork (TM) cells. The present study also provides criteria fordetermining the efficacy of interfering RNA on mRNA levels when QPCRprimers are used for measurement.

Transfection of a transformed human TM cell line designated GTM3 orHTM-3 (see Pang, I. H. et al., 1994. Curr. Eye Res. 13:51-63) wasaccomplished using standard in vitro concentrations of CTGF interferingRNA (100 nM) and LIPOFECTAMINE™ 2000 (Invitrogen, Carlsbad, Calif.) at a1:1 (w/v) ratio. A pool of commercially designed interfering RNAs ofunknown sequence (siGENOME SMARTPOOL® CTGF interfering RNA (designatedsiRNA S4 herein), Dharmacon, Lafayette, Colo.) was used to target CTGF.Scrambled and lamin A/C siRNA (Dharmacon) were used as controls.

Control experiments resulted in close to 90% knock-down efficiency oflamin A/C using lamin A/C interfering RNA when compared to the scrambledinterfering RNA control. Initial studies showed an efficiency ofknock-down of CTGF of about 20-30% when using siGENOME SMARTPOOL® CTGFsiRNA M-012633-00-0020 (siRNA S4) using primer/probe set Q2 directed tothe CTGF mRNA 3′UTR in exon 5 (FIG. 2B). Q2 is a QPCR TAQMAN®primer/probe sets from ABI (Applied Biosystems, Foster City, Calif.).

To determine the reason for the poor CTGF siRNA efficacy, severalvariables were tested. Dose response with the CTGF interfering RNA wastested to determine if a suboptimal interfering RNA concentration or asuboptimal interfering RNA:lipid ratio was being used. Resultant dataindicated poor CTGF mRNA knock-down regardless of the interfering RNAconcentration or interfering RNA:lipid ratio employed. Given theimportance of cellular uptake on siRNA activity and the inherentdifficulty of transfecting TM cells, the TM cell transfection efficiencywas determined under the above-cited conditions. Transfection efficiencywas examined as a reflection of either cell death induced by SITOX™(Dharmacon) delivery to the cell cytoplasm or cell fluorescence asmeasured by cytoplasmic fluorescence with SIGLO™ (Dharmacon). In bothcases, nearly all cells were either dead (FIG. 1A; SITOX™) orfluorescent (FIG. 1B; SIGLO™), suggesting that the transfectionefficiency was nearly quantitative and not the rate-limiting step in theprocess.

Further, three additional individual CTGF siRNA sequences from AmbionInc. (Austin, Tex.) designated siRNA S1, S2, and S3 were tested incombination with two different QPCR TAQMAN® primer/probe sets designatedQ2 and Q1 (ABI, Applied Biosystems, Foster City, Calif.). The targetsequences for Ambion siRNAs are as follows using GenBank referencesequence number NM_(—)001901 for nucleotides (nts) of CTGF:

target for S1: (nts 379-397): gggcctcttctgtgacttc SEQ ID NO: 2 targetfor S2: (nts 901-919): gggcaaaaagtgcatccgt SEQ ID NO: 5 target for S3:(nts 1488-1506): ggttagtatcatcagatag SEQ ID NO: 18

Double stranded siRNA with a 3′TT overhang on each strand for each ofthe above targeted sequences are:

siRNA S1: 5′-gggccucuucugugacuucTT-3′ SEQ ID NO: 303′-Ttcccggagaagacacugaag-5′ SEQ ID NO: 31 siRNA S2:5′-gggcaaaaagugcauccguTT-3′ SEQ ID NO: 32 3′-TTcccguuuuucacguaggca-5′SEQ ID NO: 33 siRNA S3: 5′-gguuaguaucaucagauagTT-3′ SEQ ID NO: 273′-TTccaaucauaguagucuauc-5′ SEQ ID NO: 28

The QPCR Q1 primer is a proprietary sequence from ABI ASSAY ON DEMAND™Hs00170014 ml (Applied BioSystems).

The QPCR Q2 forward primer has the sequence:

5′-CAGCTCTGACATTCTGATTCGAA-3′ SEQ ID NO: 34

and the Q2 reverse primer has the sequence:

5′-TGCCACAAGCTGTCCAGTCT-3′ SEQ ID NO: 35

The Q2 probe has the sequence:

5′-AATCGACAGGATTCCGATTCCTGAACAGTG-3′ SEQ ID NO:36 and has an FAM groupat the 5′ end (6-carboxyfluorescein) and a TAMRA group at the 3′ end(Applied Biosystems).

The location of the primer/probe sets in relation to the siRNA targetsites for the individual siRNAs is shown in FIG. 2A. Also shown in theschematic of FIG. 2A are the CTGF gene exon (boxes) and intron (lines)structure and location of siRNAs S1, S2, and S3 and QPCR primer/probesets Q1 and Q2 in relation to the GenBank CTGF sequence NM_(—)001901,the sequence of which is provided as SEQ ID NO:1.

FIG. 2B shows QPCR amplification of CTGF mRNA using the exon 5primer/probe set Q2 and siRNAs S1-S4. Using the S1 and S4 siRNAs, nosignificant knock-down of the CTGF mRNA levels was detected with the Q2primer/probe set. Knock-down was demonstrated by siRNAs S2 and S3. Theprimer/probe set Q2 has closer proximity to the targets of the S2 and S3siRNAs as compared to the target of the S1 siRNA.

FIG. 2C shows QPCR amplification of CTGF mRNA using the exon 4/5spanning primer/probe set Q. Knock-down of CTGF mRNA was demonstrated byeach of the siRNAs and an ˜90% knock-down was observed with the S2 siRNAusing the Q1 primer/probe set for detection. The primer/probe set Q1appears to be more efficient at demonstrating knock-down by the siRNAsas compared to the Q2 primer probe set.

The data of FIG. 2B and FIG. 2C suggest that the particular regionamplified using the 3′-UTR-directed primer/probe set Q2 may berelatively stable and thus a poor choice for assessing the cleavage anddegradation of the CTGF mRNA by the targeting siRNA. Therefore, siRNAefficacy may be underreported in specific cases where the QPCRamplification region lies outside the siRNA targeting region.

To reduce the chance of non-specific, off-target effects, the lowestpossible siRNA concentration for inhibiting CTGF mRNA expression wasdetermined. CTGF mRNA knock-down was assessed by QPCR amplificationusing primer/probe set Q1. A dose response of CTGF S2 siRNA in GTM3cells is shown in FIG. 3. An IC₅₀ of ˜2.5 nM was observed in GTM3 cellsafter 24 hour treatment with 0, 1, 3, 10, 30, and 100 nM dose range ofS2 siRNA. Data were fitted using GraphPad Prism 4 software (GraphPadSoftware, Inc., San Diego, Calif.) with a variable slope, sigmoidal doseresponse algorithm and a top constraint of 100%.

The results of this example demonstrate that i) trabecular meshworkcells carry out siRNA silencing, ii) all of the siRNAs cited hereineffect a degree of silencing, and iii) when using a PCR-based approachto determine the efficacy of siRNA knock-down, the PCR amplificationprimers are designed to encompass the siRNA targeting sequence foroptimum detection of silencing.

Cleavage of target mRNA by the RISC endonuclease has been shown to occurnear the center of the siRNA targeting sequence (Elbashir, S. M., etal., 2001. Genes Dev 15:188-200) and is accomplished by Argonaute RNaseHactivity (Liu, J., et al., 2004. Science 305:1437-1441). However,complete degradation of the remaining mRNA appears not to be guaranteed.Stable fragments of mRNA may remain following Argonaute cleavage andamplification of one of these fragments by QPCR may underreport thesiRNA efficacy as shown herein. The present invention provides anembodiment where QPCR primer sets encompass the siRNA target sequence toensure optimum siRNA efficiency readout.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated by reference.

Those of skill in the art, in light of the present disclosure, willappreciate that obvious modifications of the embodiments disclosedherein can be made without departing from the spirit and scope of theinvention. All of the embodiments disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. The full scope of the invention is set out in the disclosureand equivalent embodiments thereof. The specification should not beconstrued to unduly narrow the full scope of protection to which thepresent invention is entitled.

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one”, “at least one” or “one or more”.

1. A method of attenuating expression of connective tissue growth factormRNA in an eye of a subject, comprising: administering to the eye of thesubject a composition comprising an effective amount of interfering RNAhaving a length of 19 to 49 nucleotides and a pharmaceuticallyacceptable carrier, the interfering RNA comprising: a sense nucleotidesequence, an antisense nucleotide sequence, and a region of at leastnear-perfect contiguous complementarity of at least 19 nucleotides;wherein the antisense sequence hybridizes under physiological conditionsto a portion of mRNA corresponding to SEQ ID NO:1, and has a region ofat least near-perfect contiguous complementarity of at least 19nucleotides with the hybridizing portion of mRNA corresponding to SEQ IDNO:1, wherein the expression of connective tissue growth factor mRNA isattenuated.
 2. The method of claim 1 wherein the subject has aconnective tissue growth factor-associated ocular disorder.
 3. Themethod of claim 1 wherein the subject is at risk of developing aconnective tissue growth factor-associated ocular disorder.
 4. Themethod of claim 2 wherein the connective tissue growth factor-associatedocular disorder is glaucoma, macular degeneration, diabetic retinopathy,choroidal neovascularization, proliferative vitreoretinopathy or woundhealing.
 5. The method of claim 1 wherein the antisense sequence has aregion of at least near-perfect contiguous complementarity of at least21 to 23 nucleotides with the hybridizing portion of mRNA correspondingto SEQ ID NO:1 and comprises an additional TT sequence at the 3′ end ofeach of the sense and the antisense sequence.
 6. The method of claim 1wherein the sense nucleotide sequence and the antisense nucleotidesequence are connected by a loop nucleotide sequence.
 7. The method ofclaim 1 wherein the composition is administered via a topical,intravitreal, or transcleral route.
 8. The method of claim 1 wherein theantisense sequence is designed to target a nucleotide sequence of mRNAcorresponding to SEQ ID NO:1 beginning at nucleotide 379, 691, 801, 901,932, 937, 969, 986, 1119, 1170, 1201, 1346, 1473, 1478, 1481, 1488,1626, 1660, or
 1666. 9. The method of claim 1 wherein the antisensesequence is designed to target a nucleotide sequence of mRNAcorresponding to SEQ ID NO:1 beginning at nucleotide 379, 901, or 1488.10. The method of claim 1 wherein the antisense sequence is designed totarget a nucleotide sequence of mRNA corresponding to SEQ ID NO:1comprising nucleotide 379, 691, 801, 901, 932, 937, 969, 986, 1119,1170, 1201, 1346, 1473, 1478, 1481, 1488, 1626, 1660, or
 1666. 11. Themethod of claim 1 wherein the antisense sequence is designed to target anucleotide sequence of mRNA corresponding to SEQ ID NO:1 comprisingnucleotide 379, 901, or
 1488. 12. The method of claim 1 wherein theantisense sequence comprises: 3′-TTcccguuuuucacguaggca-5′. SEQ ID NO: 33


13. The method of claim 1 wherein the antisense sequence comprises:3′-TTcccggagaagacacugaag-5′. SEQ ID NO: 31


14. The method of claim 1 wherein the antisense sequence comprises:3′-TTccaaucauaguagucuauc-5′. SEQ ID NO: 28


15. The method of claim 1 wherein the interfering RNA comprises:5′-gggccucuucugugacuucTT-3′ SEQ ID NO: 30 and3′-TTcccggagaagacacugaag-5′. SEQ ID NO: 31


16. The method of claim 1 wherein the interfering RNA comprises:5 -gguuaguaucaucagauagTT-3′ SEQ ID NO: 27 and3′-TTccaaucauaguagucuauc-5′. SEQ ID NO: 28


17. (canceled)
 18. The method of claim 1 further comprisingadministering to the eye of the subject a second interfering RNA havinga length of 19 to 49 nucleotides, and comprising: a sense nucleotidesequence, an antisense nucleotide sequence, and a region of at leastnear-perfect complementarity of at least 19 nucleotides; wherein theantisense sequence of the second interfering RNA hybridizes underphysiological conditions to a second portion of mRNA corresponding toSEQ ID NO:1, and the antisense sequence has a region of at leastnear-perfect contiguous complementarity of at least 19 nucleotides withthe second hybridizing portion of mRNA corresponding to SEQ ID NO:1. 19.A method of attenuating expression of connective tissue growth factormRNA in an eye of a subject, comprising: administering to the eye of thesubject a composition comprising an effective amount of single-strandedinterfering RNA having a length of 19 to 49 nucleotides and apharmaceutically acceptable carrier, wherein the single strandedinterfering RNA hybridizes under physiological conditions to a portionof mRNA corresponding to SEQ ID NO:1 beginning at nucleotide 379, 691,801, 901, 932, 937, 969, 986, 1119, 1170, 1201, 1346, 1473, 1478, 1481,1488, 1626, 1660, or 1666, and the interfering RNA has a region of atleast near-perfect complementarity with the hybridizing portion of mRNAcorresponding to SEQ ID NO:1, wherein the expression of connectivetissue growth factor mRNA is attenuated. 20-36. (canceled)